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Abstract

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Materials

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Developmental Biology

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Published: October 8th, 2016

DOI:

10.3791/54252

1Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 2Department of Bioengineering and Orthopaedic Surgery, University of Pittsburgh, 3Research Laboratory of Electronics and Department of Biological Engineering, Massachusetts Institute of Technology, 4MRC Centre for Regenerative Medicine, University of Edinburgh, 5Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, 6Department of Orthopaedic Surgery, University of Texas Health Science Center at Houston, 7Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital, David Geffen School of Medicine, University of California at Los Angeles

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

The heart has long been considered a post-mitotic organ. However, recent studies have demonstrated the presence of limited cardiomyocyte turnover in adult human hearts1. Native stem/progenitor cells with cardiomyocyte differentiation potential have also been identified within the myocardium in adult rodent and human hearts, including Sca-1+, c-kit+, cardiosphere-forming, and most recently, perivascular precursor cells2,3. These cells represent attractive candidates for therapies aimed at enhancing cardiac repair/regeneration through cell transplantation or stimulation of in-situ proliferation.

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1. Processing of Human Cardiac Sample

  1. Ensure that all fluids, containers, instruments, and the dedicated operational area are sterile.
  2. Place the cardiac tissue sample (procured by the tissue bank or surgical team) in storage medium composed of chilled Dulbecco's modified Eagle medium (DMEM) containing 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) on ice for transportation3.
  3. Remove the cardiac sample from the storage medium and wash with a washing medium c.......

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Single cells were distinguished from debris and doublets on the basis of forward and side scatter distributions. Live cells were identified by their failure to take up the DAPI dye. The gating strategy was chosen on the basis of isotype control labelling of this live, whole-cardiac cell dissociation (Figure 1). From the live cells, CD45+ cells were first gated out, followed by CD56+ cells. CD144+ endothelial cells were then removed from th.......

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Increasing evidence supports a limited regenerative capacity of the adult human heart after injury. Identification and characterization of native precursor cells responsible for such regenerative responses in injured hearts are critical for both the understanding of associated mechanisms and signalling pathways and the development of approaches to utilize these cells therapeutically.

Previous protocols have described the isolation of perivascular precursor cell subsets from human skeletal musc.......

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The authors wish to thank Shonna Johnston, Claire Cryer, Fiona Rossi and Will Ramsay at the University of Edinburgh and Alison Logar and Megan Blanchard at the University of Pittsburgh for their expert assistance with flow cytometry. We also wish to thank Anne Saunderson and Lindsay Mock for their help with obtaining human tissues. Human adult and fetal heart tissue samples were procured with full ethics permission of the NHS Scotland Tayside Committee on Medical Research Ethics and the NHS Lothian Research Ethics Committee (REC08/S1101/1) respectively. This work was supported by grants from the Medical Research Council (BP), British Heart Foundation (BP), Commonwealt....

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Name Company Catalog Number Comments
AbC Anti-mouse Bead Kit Molecular Probes A-10344
Collagenase I Gibco 17100-017 Reconstitute powder as required and filter sterilise
Collagenase II Gibco 17101-015
Collagenase IV Gibco 17104-019
anti-human CD34-PE BD Pharmingen 555822 Keep sterile
anti-human CD45-APC-Cy7 BD Pharmingen 557833 Keep sterile
anti-human CD56-PE-Cy7 BD Pharmingen 557747 Keep sterile
anti-human CD144-PerCP-Cy5.5 BD Pharmingen 561566 Keep sterile
anti-human CD146-AF647 AbD Serotec MCA2141A647 Keep sterile
EGM2-BulletKit Lonza CC-3162 For collection of cells and culture until adhered
DMEM, high glucose, GlutaMAX without sodium pyruvate ThermoFischer Scientific 10566-016
Fetal Bovine Serum ThermoFischer Scientific 10500-064 Freeze in aliquots and keep sterile
Gelatin Sigma Aldrich G1393 Dilute with sterile water
IgG1k-PE BD Pharmingen 559320 Keep sterile
IgG1k-APC-Cy7 BD Pharmingen 557873 Keep sterile
IgG1k-PE-Cy7 BD Pharmingen 557872 Keep sterile
IgG1k-PerCP-Cy5.5 BD Pharmingen 561566 Keep sterile
IgG1k-647 AbD Serotec MCA1209A647 Keep sterile
Mouse serum Sigma Aldrich M5905 Keep sterile
Paraffin Film - Parafilm M Sigma Aldrich P7793
Penicillin-Streptomycin Gibco 15979-063 Freeze in aliquots and keep sterile
Phosphate buffered saline pH 7.4 ThermoFischer Scientific 10010-023 Keep sterile
Red Blood Cell Lysing Buffer Hybri-Max Sigma Aldrich R7757 Keep sterile
Trypan Blue Solution Sigma Aldrich T8154
Trypsin-EDTA 0.5%(10X) Invitrogen 15400-054
 FACSARIA FUSION BD Pharmingen Fluorescence Activated Cell Sorter

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